Unveiling the inverse antimicrobial impact of a hetero-chitooligosaccharide on Candida tropicalis growth and biofilm formation.

Chitosan and chitooligosaccharide (COS) are renowned for their potent antimicrobial prowess, yet the precise antimicrobial efficacy of COS remains elusive due to scant structural information about the utilized saccharides. This study delves into the antimicrobial potential of COS, spotlighting a distinct hetero-chitooligosaccharide dubbed DACOS. In contrast to other COS, DACOS remarkably fosters the growth of Candida tropicalis planktonic cells and fungal biofilms. Employing gradient alcohol precipitation, DACOS was fractionated, unveiling diverse structural characteristics and differential impacts on C. tropicalis. Notably, in a murine model of systemic candidiasis, DACOS, particularly its 70 % alcohol precipitates, manifests a promotive effect on Candida infection. This research unveils a new pathway for exploring the intricate nexus between the structural attributes of chitosan oligosaccharides and their physiological repercussions, underscoring the imperative of crafting chitosan and COS with meticulously defined structural configurations.

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

Production of CA125 with Tn-antigens using a glycosylphosphatidylinositol anchoring system.

Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn-antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn-antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn-antigen showed a significant increase, whereas the expression of the T-antigen (galactose-ß1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn-antigen modification.

Comprehensive in silico analysis of glycosylphosphatidylinositol- anchored protein (GPI-AP) related genes expression profiles in human normal and cancer tissues.

In human cells, there are more than 146 glycosylphosphatidylinositol-anchored proteins (GPI-APs), including receptors, ligands, adhesion molecules and enzymes. The proteins are associated with membrane microdomains called lipid rafts through GPI, and plays a variety of important biological functions. At present, plenty of studies have been carried out on the biosynthesis of GPI-APs. The biosynthesis of GPI-APs requires at least 20 steps, and more than 40 GPI biosynthetic genes have been identified. However, it remains unclear how expression of GPI-AP related genes is regulated in normal and cancer tissues. In this study, we utilized gene expression data from both the TCGA database and GTEx portal to analysis the gene expression involved in GPI-AP biosynthesis and encoding GPI-APs in normal and cancer tissues. In order to perform a comprehensive analysis, we employed the GlycoMaple, a tool that is specifically designed to analyze glycosylation pathways. The results showed that compared with normal tissues, the expression of genes involved in GPI-AP biosynthesis in cancer tissues such as early glioma, glioblastoma multiforme, pancreatic cancer, testicular germ cell carcinoma, skin primary cutaneous melanoma and skin metastatic cutaneous melanoma, was changed significantly. Particularly, the expression of PIGY in these six cancers was increased. In addition, the expression of CD14, a GPI-AP gene, was increased in these six cancers. The expression of GAS1, GPC2 and GPC4 was increased only in early glioma and glioblastoma multiforme indicating that some GPI-APs such as GAS1 can be used as biomarkers of glioma. This study provides new insights into the expression of GPI-AP related genes in normal and cancer tissues, and lays a solid foundation for the development of GPI-APs as biomarkers.

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins.

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.

Comprehensive proteome, phosphoproteome and kinome characterization of luminal A breast cancer.

Background: Breast cancer is one of the most frequently occurring malignant cancers worldwide. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two most common histological subtypes of breast cancer. In this study, we aimed to deeply explore molecular characteristics and the relationship between IDC and ILC subtypes in luminal A subgroup of breast cancer using comprehensive proteomics and phosphoproteomics analysis. Methods: Cancer tissues and noncancerous adjacent tissues (NATs) with the luminal A subtype (ER- and PR-positive, HER2-negative) were obtained from paired IDC and ILC patients respectively. Label-free quantitative proteomics and phosphoproteomics methods were used to detect differential proteins and the phosphorylation status between 10 paired breast cancer and NATs. Then, the difference in protein expression and its phosphorylation between IDC and ILC subtypes were explored. Meanwhile, the activation of kinases and their substrates was also revealed by Kinase-Substrate Enrichment Analysis (KSEA). Results: In the luminal A breast cancer, a total of 5,044 high-confidence proteins and 3,808 phosphoproteins were identified from 10 paired tissues. The protein phosphorylation level in ILC tissues was higher than that in IDC tissues. Histone H1.10 was significantly increased in IDC but decreased in ILC, Conversely, complement C4-B and Crk-like protein were significantly decreased in IDC but increased in ILC. Moreover, the increased protein expression of Septin-2, Septin-9, Heterogeneous nuclear ribonucleoprotein A1 and Kinectin but reduce of their phosphorylation could clearly distinguish IDC from ILC. In addition, IDC was primarily related to energy metabolism and MAPK pathway, while ILC was more closely involved in the AMPK and p53/p21 pathways. Furthermore, the kinomes in IDC were primarily significantly activated in the CMGC groups. Conclusions: Our research provides insights into the molecular characterization of IDC and ILC and contributes to discovering novel targets for further drug development and targeted treatment.

Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.

Simple N-Glycan Profile Analysis Using Lectin Staining, Mass Spectrometry, and GlycoMaple.

The Golgi apparatus is one of the major sites of protein and lipid glycosylation and processing. Protein N-glycosylation plays critical roles in protein folding, transport, stability, and activity. Various glycosyltransferases and glycoside hydrolases are localized at each cisterna in the Golgi apparatus and synthesize a large variety of N-glycan structures. The biosynthetic pathways of N-glycans are complicated, which hiders the rational design of glycan metabolic pathways. In addition, the analysis of glycan structure requires specialized instruments for analyses such as mass spectrometry, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy, which are not familiar to all laboratories. Here, we introduce relatively simple methods for N-glycan analysis, including disruption of genes encoding glycosyltransferases or glycoside hydrolases, glycan structural analysis using lectins and mass spectrometry, and visualization of glycan metabolic pathways in silico.

Mitotic phosphorylation inhibits the Golgi mannosidase MAN1A1.

N-glycans are processed mainly in the Golgi, and a well-organized Golgi structure is required for accurate glycosylation. However, during mitosis the Golgi undergoes severe fragmentation. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes. It is unclear how cells avoid glycosylation defects during mitosis. In this study, we report that Golgi α-1,2-mannosidase IA (MAN1A1), the first enzyme that cargo proteins encounter once arriving the Golgi, is phosphorylated at serine 12 by CDK1 in mitosis, which attenuates its activity, affects the production of glycan isomers, and reduces its interaction with the subsequent glycosyltransferase, MGAT1. Expression of wild-type MAN1A1, but not its phosphomimetic mutant, rescues the glycosylation defects in mannosidase I-deficient cells, whereas expression of its phosphorylation-deficient mutant in mitosis increases the formation of complex glycans. Our study reveals that glycosylation is regulated by cytosolic signaling during the cell cycle.

Proteome and Glycoproteome Analyses Reveal the Protein N-Linked Glycosylation Specificity of STT3A and STT3B.

STT3A and STT3B are the main catalytic subunits of the oligosaccharyltransferase complex (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and post-translocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomic and glycoproteomic analyses in the gene knock-out (KO) and wild-type HEK293 cells. In total, 3961 proteins, 4265 unique N-linked intact glycopeptides and 629 glycosites representing 349 glycoproteins were identified from all these cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, total mannosylated N-glycans were reduced and fucosylated N-glycans were increased in STT3A-KO cells, which were caused by the differential expression of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells, and the hyperglycosylation of ENPL was caused by the endoplasmic reticulum (ER) stress due to the STT3A deletion. Furthermore, the increased expression of the ATF6 and PERK indicated that the unfolded protein response also happened in STT3A-KO cells. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.

Receptor for advanced glycation end-products (RAGE) mediates phagocytosis in nonprofessional phagocytes.

In mammals, both professional phagocytes and nonprofessional phagocytes (NPPs) can perform phagocytosis. However, limited targets are phagocytosed by NPPs, and thus, the mechanism remains unclear. We find that spores of the yeast Saccharomyces cerevisiae are internalized efficiently by NPPs. Analyses of this phenomenon reveals that RNA fragments derived from cytosolic RNA species are attached to the spore wall, and these fragments serve as ligands to induce spore internalization. Furthermore, we show that a multiligand receptor, RAGE (receptor for advanced glycation end-products), mediates phagocytosis in NPPs. RAGE-mediated phagocytosis is not uniquely induced by spores but is an intrinsic mechanism by which NPPs internalize macromolecules containing RAGE ligands. In fact, artificial particles labeled with polynucleotides, HMGB1, or histone (but not bovine serum albumin) are internalized in NPPs. Our findings provide insight into the molecular basis of phagocytosis by NPPs, a process by which a variety of macromolecules are targeted for internalization.

Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.

SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.

C18orf32 loss-of-function is associated with a neurodevelopmental disorder with hypotonia and contractures.

Glycosylphosphatidylinositol (GPI) functions to anchor certain proteins to the cell surface. Although defects in GPI biosynthesis can result in a wide range of phenotypes, most affected patients present with neurological abnormalities and their diseases are grouped as inherited-GPI deficiency disorders. We present two siblings with global developmental delay, brain anomalies, hypotonia, and contractures. Exome sequencing revealed a homozygous variant, NM_001035005.4:c.90dupC (p.Phe31Leufs*3) in C18orf32, a gene not previously associated with any disease in humans. The encoded protein is known to be important for GPI-inositol deacylation. Knockout of C18orf32 in HEK293 cells followed by a transfection rescue assay revealed that the PIPLC (Phosphatidylinositol-Specific Phospholipase C) sensitivity of GPI-APs (GPI-anchored proteins) was restored only by the wild type and not the mutant C18orf32. Immunofluorescence revealed that the mutant C18orf32 was localized to the endoplasmic reticulum and was also found as aggregates in the nucleus. In conclusion, we identified a pathogenic variant in C18orf32 as the cause of a novel autosomal recessive neurodevelopmental disorder with hypotonia and contractures. Our results demonstrate the importance of C18orf32 in the biosynthesis of GPI-anchors, the molecular impact of the variant on the protein function, and add a novel candidate gene to the existing repertoire of genes implicated in neurodevelopmental disorders.

Hydrophobicity of the C-terminal GPI-anchor Attachment Signals Determines ER-associated Degradation Pathway of Precursor Proteins / 中国生物化学与分子生物学报

More than 150 glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are expressed in mammalian cells and involved in various physiological processes such as immune recognition, cell communication and signal transduction. GPI is transferred to proteins in the endoplasmic reticulum (ER). When GPI-anchoring is impaired, precursor proteins are thought to be degraded through ER-associated degradation (ERAD). However, the mechanism of their degradation in ERAD remains unclear. To investigate the impact of ERAD pathways on degradation of GPI precursor proteins, we used series of knockout (KO) human embryonic kidney 293 (HEK293) cells defective in PIGS gene, which encodes a GPI transamidase complex subunit, combined with KO in HRD1 (PIGS-HRD1-KO) or GP78 (PIGSGP78-KO), which encodes the E3 ubiquitin ligases for the ERAD pathways. We compared the stability of 16 GPI precursor proteins in the ERAD-deficient cells with the parental PIGS-KO cells. Western blotting data showed that the GPI precursor proteins were stabilized in either PIGS-HRD1-KO (I

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit.

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Novel Insight Into Glycosaminoglycan Biosynthesis Based on Gene Expression Profiles.

Glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate, except for hyaluronan that is a free polysaccharide, are covalently attached to core proteins to form proteoglycans. More than 50 gene products are involved in the biosynthesis of GAGs. We recently developed a comprehensive glycosylation mapping tool, GlycoMaple, for visualization and estimation of glycan structures based on gene expression profiles. Using this tool, the expression levels of GAG biosynthetic genes were analyzed in various human tissues as well as tumor tissues. In brain and pancreatic tumors, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be upregulated. In breast cancerous tissues, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be up- and down-regulated, respectively, which are consistent with biochemical findings published in the literature. In addition, the expression levels of the chondroitin sulfate-proteoglycan versican and the dermatan sulfate-proteoglycan decorin were up- and down-regulated, respectively. These findings may provide new insight into GAG profiles in various human diseases including cancerous tumors as well as neurodegenerative disease using GlycoMaple analysis.

A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins.

Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.

Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition.

A FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface. In the screening, SLC35B2, which encodes 3'-phosphoadenosine-5'-phosphosulfate transporter 1, was identified as the candidate gene. The detection of FLAG-tagged misfolded proteins at the cell surface was significantly increased in SLC35B2-knockout cells. Furthermore, protein tyrosine sulfation mediated by tyrosyl-protein sulfotransferase 2 (TPST2) suppressed FLAG-tagged protein detection. Localization analysis of the FLAG-tagged misfolded proteins confirmed that defects in tyrosine sulfation are only responsible for enhancing anti-FLAG staining on the plasma membrane but not inducing the localization change of misfolded proteins on the plasma membrane. These results suggest that a FLAG tag on the misfolded protein would be sulfated, causing a reduced detection by the M2 anti-FLAG antibody. Attention should be required when quantifying the FLAG-tagged proteins in the secretory pathway.

Cell engineering for the production of hybrid-type N-glycans in HEK293 cells.

Glycoprotein therapeutics are among the leading products in the biopharmaceutical industry. The heterogeneity of glycans in therapeutic proteins is an issue for maintaining quality, activity and safety during bioprocessing. In this study, we knocked out genes encoding Golgi α-mannosidase-II, MAN2A1 and MAN2A2 in human embryonic kidney 293 (HEK293) cells, establishing an M2D-KO cell line that can produce recombinant proteins mainly with hybrid-type N-glycans. Furthermore, FUT8, which encodes α1,6-fucosyltransferase, was knocked out in the M2D-KO cell line, establishing a DF-KO cell line that can express noncore fucosylated hybrid-type N-glycans. Two recombinant proteins, lysosomal acid lipase and constant fragment of human IgG1, were expressed in the M2D-KO and DF-KO cell lines. Glycan structural analysis revealed that complex-type N-glycans were removed in both M2D-KO and DF-KO cells. Our results suggest that these cell lines are suitable for the production of therapeutic proteins with hybrid-type N-glycans. Moreover, KO cell lines would be useful as models for researching the mechanism of antimetastatic effects in human tumours by swainsonine treatment.